Repair of larynx, trachea, and other fibrocartilaginous tissues

ABSTRACT

Provided herein are methods and devices for inducing the formation of functional replacement nonarticular cartilage tissues and ligament tissues. These methods and devices involve the use of osteogenic proteins, and are useful in repairing defects in the larynx, trachea, interarticular menisci, intervertebral discs, ear, nose, ribs and other fibrocartilaginous tissues in a mammal.

FIELD OF THE INVENTION

This invention relates to the field of ligament and nonarticularcartilage tissue repair using osteogenic proteins.

BACKGROUND OF THE INVENTION

Osteogenic and chondrogenic proteins are able to induce theproliferation and differentiation of progenitor cells into functionalbone, cartilage, tendon, and/or ligamentous tissue. These proteins,referred to herein as “osteogenic proteins,” “morphogenic proteins” or“morphogens,” include members of the bone morphogenetic protein (“BMP”)family identified by their ability to induce endochondral bonemorphogenesis. The osteogenic proteins generally are classified in theart as a subgroup of the TGF-β superfamily of growth factors. Hogan,Genes & Development 10:1580-1594 (1996). Osteogenic proteins include themammalian osteogenic protein-1 (OP-1, also known as BMP-7) and itsDrosophila homolog 60 Å, osteogenic protein-2 (OP-2, also known asBMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A orCBMP-2A) and its Drosophila homolog DPP, BMP-3, BMP-4 (also known asBMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9,BMP-10, BMP-11, BMP-12, GDF-3 (also known as Vgr2), GDF-8, GDF-9,GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known asCDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known asCDMP-3), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, andNEURAL.

Osteogenic proteins typically include secretory peptides sharing commonstructural features. Processed from a precursor “pro-form,” the matureform of an osteogenic protein is a disulfided-bonded homo- orhetero-dimer, with each subunit having a carboxyl terminal activedomain. This domain has approximately 97-106 amino acid residues andcontains a conserved pattern of cysteine residues. See, e.g., Massague,Annu. Rev. Cell Biol. 6:597 (1990); Sampath et al., J. Biol. Chem.265:13198 (1990).

Osteogenic proteins can stimulate the proliferation and differentiationof progenitor cells when administered with an appropriate matrix orsubstrate to a mammal. As a result, they can induce bone formation,including endochondral bone formation, under conditions where truereplacement bone would not otherwise occur. For example, when combinedwith a matrix material, osteogenic proteins induce formation of new bonein large segmental bone defects, spinal fusions, and fractures.

The larynx extends from the tongue to the trachea. The trachea is acartilaginous and membranous tube extending from the lower end of thelarynx to its division into the two principal bronchi.Fibrocartilaginous tissue is found in the larynx. Cartilage forms theskeletal framework of the larynx and is interconnected by ligaments andfibrous membranes. The hyoid bone is intimately associated with thelarynx, although it is usually regarded as a separate structure with adistinct function.

Abnormalities of the laryngeal skeleton influence its respiratory,defensive and phonatory functions, and can result in suffocation or lossof voice. Abnormalities can be congenital, such as cleft larynx, oracquired, such as an edema of the glottis. Excessive ossification of oneor more hyaline cartilage tissues also may limit the respiratory orphonatory function. Still other abnormalities include ulceration of thelarynx as a result of disease, e.g., syphilis, tuberculosis ormalignancy. Abnormalities also can result from mechanical trauma to thelarynx or trachea, including complications from tracheotomies. Severaldiseases of the human larynx, including laryngeal cancer, involve thelaryngeal skeleton. Treatment of these and other conditions may involvepartial or complete removal of the laryngeal skeleton or trachea(tracheotomy, laryngotomy, or laryngotracheotomy). Surgicalreconstructive procedures of the larynx or trachea are complex. To date,reconstruction has relied on cartilage grafts, small intestine grafts,and cellular adhesives such as fibrinogen or cyanoacrylate to reattachtorn tissue. Common complications include graft rejection and/or fibroustransformation of autografts or allografts.

Fibrocartilaginous tissue is found not only in the larynx, but also inother regions including the ear, nose, ribs, intervertebral discs andinterarticular menisci. Repair and reconstruction of defects in thesetissues requires regeneration of appropriate functional replacementfibrocartilage.

SUMMARY OF THE INVENTION

The present invention provides methods and devices for inducing in vivoformation of functional (e.g., mechanically acceptable) replacementnonarticular cartilage and ligament tissues.

In a method of the invention, an osteogenic protein is provided in abiocompatible, bioresorbable carrier to a defect locus in a nonarticularcartilage tissue of a mammal, thereby inducing the formation offunctional replacement cartilage tissue. The defect locus can be in thelarynx, trachea, intervertebral discs, interarticular menisci, ear,nose, ribs, or other fibrocartilaginous tissues of the mammal. Forinstance, the method can be used to repair defects in cricoid, thyroid,arytenoid, cuneiform, corniculate and epiglottic cartilage tissues, aswell as any other nonarticular hyaline cartilage tissues. Under certaincircumstances, the osteogenic protein and the carrier are preferablyplaced under the perichondrium of the target tissue.

The carrier used in this invention is biocompatible in that it is nottoxic and does not elicit severe inflammatory reactions in the body. Thecarrier is also bioresorbable in that it can be at least partially, andpreferably entirely, resorbed at the repaired locus within a clinicallyacceptable period of time, e.g., 4 months to a year. The carrier caninclude a matrix or “scaffold” structure, or it can be substantiallymatrix-free. The carrier may be solid (e.g., porous or particulate), orin a gel, paste, liquid or other injectable form. Suitable carrierscontain materials that include, but are not limited to, allogenic tissue(e.g., devitalized allogenic, autologous, or xenogenic cartilagetissue), collagen (e.g., Types I and II collagen), celluloses (e.g.,alkylcelluloses such as carboxymethylcellulose), calcium phosphates(e.g., hydroxyapatite), poloxamers (e.g., PLURONIC F127), gelatins,polyethylene glycols (e.g., PEG 3350), dextrins, vegetable oils (e.g.,sesame oil), and polymers comprised of lactic acid, butyric acid, and/orglycolic acid. Autologous or autogenic blood can also be included in thecarrier, because it has been found that such inclusion speeds up thehealing process.

Also embraced within this invention are implantable devices forrepairing nonarticular cartilage tissues or ligament tissues. Suchdevices contain one or more osteogenic proteins disposed in a carriercontaining, e.g., devitalized cartilage, Type I collagen, orcarboxymethylcellulose.

This invention also provides a method of promoting chondrogenesis at adefect locus in a mammal. In this method, an osteogenic protein isprovided to a devitalized cartilage carrier to the defect locus, whereinthe cartilage has been configured to fit into the defect locus.

Osteogenic proteins useful in this invention include, but are notlimited to, OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6,BMP-9, BMP-10, BMP-1, BMP-12, BMP-13, BMP-14, BMP-15, GDF-1, GDF-2,GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12,CDMP-1, CDMP-2, CDMP-3, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN,SCREW, ADMP, NEURAL, and TGF-β. As used herein, the terms “morphogen,”“bone morphogen,” “BMP,” “osteogenic protein” and “osteogenic factor”embrace the class of proteins typified by human osteogenic protein 1(hOP-1).

One of the preferred osteogenic proteins is OP-1. Nucleotide and aminoacid sequences for hOP-1 are provided in SEQ ID NOs:1 and 2,respectively. For ease of description, hOP-1 is recited as arepresentative osteogenic protein. It will be appreciated by theordinarily-skilled artisan, however, that OP-1 is merely representativeof a family of morphogens.

This family of morphogens include biologically active variants of any ofthe above-listed proteins, including variants containing conservativeamino acid changes; and osteogenically active proteins having theconserved seven-cysteine skeleton or domain as defined below. Forinstance useful osteogenic proteins also include those containingsequences that share at least 70% amino acid sequence homology with theC-terminal seven-cysteine domain of hOP-1, which domain corresponds tothe C-terminal 102-106 amino acid residues of SEQ ID NO:2.

To determine the percent homology of a candidate amino acid sequence tothat seven-cysteine domain, the candidate sequence and the sequence ofthe domain are aligned. The alignment can be made with, e.g., thedynamic programming algorithm described in Needleman et al., J. Mol.Biol. 48:443 (1970), and the Align Program, a commercial softwarepackage produced by DNAstar, Inc. The teachings by both sources areincorporated by reference herein. An initial alignment can be refined bycomparison to a multi-sequence alignment of a family of relatedproteins. Once the alignment between the candidate sequence and theseven-cysteine domain is made and refined, a percent homology score iscalculated. The aligned amino acid residues of the two sequences arecompared sequentially for their similarity to each other. Similarityfactors include similar size, shape and electrical charge. Oneparticularly preferred method of determining amino acid similarities isthe PAM250 matrix described in Dayhoff et al., Atlas of Protein Sequenceand Structure 5:345-352 (1978& Supp.), herein incorporated by reference.A similarity score is first calculated as the sum of the alignedpairwise amino acid similarity scores. Insertions and deletions areignored for the purposes of percent homology and identity. Accordingly,gap penalties are not used in this calculation. The raw score is thennormalized by dividing it by the geometric mean of the scores of thecandidate sequence and the seven-cysteine domain. The geometric mean isthe square root of the product of these scores. The normalized raw scoreis the percent homology.

Useful osteogenic proteins also include those containing sequences thatshare greater than 60% identity with the seven-cysteine domain. In otherembodiments, useful osteogenic proteins are defined as osteogenicallyactive proteins having any one of the generic sequences defined herein,including OPX (SEQ ID NO:3) and Generic Sequences 7 and 8 (SEQ ID NO:4and SEQ ID NO:5, respectively), or Generic Sequences 9 and 10 (SEQ IDNO:6 and SEQ ID NO:7, respectively).

In another aspect, the instant invention provides a kit for practice ofthe above-described methods. As contemplated herein, one embodiment of akit for inducing local laryngeal or tracheal tissue formation includesan improved device wherein the osteogenic protein and carrier arepackaged in the same receptacle. In other embodiments, wetting orbinding agent(s) also are provided and packaged separately from othercomponents.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that osteogenic proteins cangenerate functional replacement nonarticular cartilage and/or ligamenttissues when provided locally at a defect site in a mammal. Suchnonarticular cartilage tissues include laryngeal, tracheal, and otherfibrocartilaginous tissues such as the tissues of intervertebral discs,ribs, skeletal interarticular menisci, the ear and the nose. Devices,kits and methods of the invention are useful in restoring lost orimpaired functions resulting from loss or injuries of these tissues in amammal, e.g., a human.

In order for the invention to be more fully understood, various types ofcartilage, cartilaginous tissues and organs are described below.Articular cartilage covers the articulating surfaces of the portions ofbones in joints. The cartilage allows movement in joints without directbone-to-bone contact, thereby preventing wearing down and damage ofopposing bone surfaces. Articular cartilage has no tendency toossification. The cartilage surface appears smooth and pearlymacroscopically, and is finely granular under high power magnification.Such cartilage is referred to as hyaline cartilage, as opposed tofibrocartilage and elastic cartilage. Articular cartilage appears toderive its nutriment partly from the vessels of the neighboring synovialmembrane, partly from those of the bone that it covers. Articularcartilage is associated with the presence of Type II and Type IXcollagen and various well-characterized proteoglycans, and with theabsence of Type X collagen, which is associated with endochondral boneformation. For a detailed description of articular cartilagemicro-structure, see, for example, Aydelotte and Kuettner, Conn. Tiss.Res. 18:205 (1988); Zanetti et al., J. Cell Biol. 101:53 (1985); andPoole et al., J. Anat. 138:13 (1984).

Other types of permanent cartilage in adult mammals includefibrocartilage and elastic cartilage. In fibrocartilage, themucopolysaccharide network is interlaced with prominent collagen bundlesand the chondrocytes are more widely scattered than in hyalinecartilage. White fibrocartilage consists of a mixture of white fibroustissue and cartilaginous tissue in various proportions. Secondarycartilaginous joints are formed by discs of fibrocartilage that joinvertebrae in the vertebral column. Interarticular fibrocartilages arefound in those joints which are most exposed to violent concussion andsubject to frequent movement, e.g., the meniscus of the knee. Examplesof such joints include the temporo-mandibular, sterno-clavicular,acromio-clavicular, wrist- and knee-joints. Such fibrocartilaginousdiscs, which adhere closely to both of the opposed surfaces, arecomposed of concentric rings of fibrous tissue, with cartilaginouslaminae interposed. An example of such fibrocartilaginous discs is theintervertebral discs of the spine. Connecting fibrocartilages areinterposed between the bony surfaces of those joints which admit of onlyslight mobility, as between the bodies of the vertebrae and between thepubic bones. Circumferential fibrocartilages surround the margin of someof the articular cavities, as the cotyloid cavity of the hip and theglenoid cavity of the shoulder, they serve to deepen the artictularsurface, and to protect its edges. Stratiform fibrocartilages refer tothe thin coating to osseous grooves through which the tendons of certainmuscles glide. Interarticular fibrocartilage is considered herein asnonarticular cartilage, so as to distinguish from articular cartilagethat consists mainly of hyaline. When present in lesser amounts, as inarticular discs, glenoid and acetabular labra, the cartilaginous liningof bony grooves for tendons and some articular cartilage, fibrocartilageis unlike other types of cartilage in having much Type I (generalconnective tissue) collagen in its matrix; it is then perhaps' bestregarded as a mingling of the two types of tissue, for example where aligament or tendinous tissue inserts into hyaline cartilage, rather thana specific type of cartilage. See, e.g., Gray's Anatomy.

Elastic cartilage contains collagen fibers that are histologicallysimilar to elastin fibers. Such cartilage is found in the human body inthe auricle of the external ear, the Eustachian tubes, the corniculalaryngis, and the epiglottis. As with all cartilage, elastic cartilagealso contains chondrocytes and a matrix, the latter being pervaded inevery direction, by a network of yellow elastic fibers, branching andanastomosing in all directions except immediately around each cell,where there is a variable amount of non-fibrillated hyaline,intercellular substance.

As used herein “cartilage” is distinct from the fibrotic cartilaginoustissues, which occur in scar tissue, for example, and are keloid andtypical of scar-type tissue, i.e., composed of capillaries and abundant,irregular, disorganized bundles of Type I and Type II collagen.

The primary laryngeal cartilages are either hyaline cartilage orfibrocartilage, particularly elastic fibrocartilage. Specifically, thecorniculate, cuneiform, tritiate and epiglottic cartilages are elasticfibrocartilage with little tendency to ossify or calcify over time. Thethyroid, cricoid and most of the arytenoid cartilage are hyalinecartilage and can undergo mottled calcification or ossification with ageand can impair the mechanical acceptability of the tissue. The primarylaryngeal ligaments include the extrinsic ligaments (e.g., thyrohoidmembrane and its component ligaments), the intrinsic ligaments (e.g.,cricothyroid membrane and its component ligaments), the vestibular foldsand associated ligaments, the vocal folds and associated ligaments.

The trachea, or windpipe, is a cartilaginous and membranous cylindricaltube, flattened posteriorly. It extends from the lower part of thelarynx, on a level with the sixth cervical vertebra, to opposite thefourth, or sometimes the fifth, dorsal vertebra, where it divides intotwo bronchi, one for each lung. The trachea is composed of imperfect,hyaline cartilaginous rings, which are completed by fibrous membrane.They are highly elastic, but sometimes become calcified in advancedlife. The cartilages are enclosed in an elastic fibrous membrane.

I. Protein Considerations

In its mature, native form, a naturally occurring osteogenic protein isa glycosylated dimer, typically having an apparent molecular weight ofabout 30-36 kD as determined by SDS-PAGE. When reduced, the 30 kDprotein gives rise to two glycosylated polypeptide subunits havingapparent molecular weights of about 16 kD and 18 kD. In the reducedstate, the protein has no detectable osteogenic activity. Theunglycosylated protein, which also has osteogenic activity, has anapparent molecular weight of about 27 kD. When reduced, the 27 kDprotein gives rise to two unglycosylated polypeptides, each having amolecular weight of about 14 kD to about 16 kD. Typically, naturallyoccurring osteogenic proteins are translated as a precursor having aN-terminal signal peptide usually less than about 30 amino acids inlength. The signal peptide is followed by a “pro” domain that is cleavedto yield the mature C-terminal domain. The signal peptide is cleavedrapidly upon translation, at a cleavage site that can be predicted in agiven sequence using the method of Von Heijne, Nucleic Acids Research14:4683-4691 (1986). The pro domain usually is about three times largerthan the fully processed mature C-terminal domain.

Osteogenic proteins useful herein include any known naturally occurringnative proteins, including allelic, phylogenetic counterparts and othervariants thereof. Useful osteogenic proteins also include those that arebiosynthetically produced (e.g., including “muteins” or “mutantproteins”) and those that are new, osteogenically active members of thegeneral morphogenic family of proteins. Particularly useful sequencesinclude those comprising the C-terminal 96 to 102 amino acid residuesof: DPP (from Drosophila), Vg-1 (from Xenopus), Vgr-1 (from mouse), theOP1 and OP2 proteins (U.S. Pat. No. 5,011,691 and Oppermann et al.), aswell as the proteins referred to as BMP-2, BMP-3, BMP-4 (WO 88/00205,U.S. Pat. No. 5,013,649 and WO 91/18098), BMP-5 and BMP-6 (WO 90/11366,PCT/US90/01630), BMP-8 and BMP-9. Other proteins useful in the practiceof the invention include active forms of OP1, OP2, OP3, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, DPP, Vg-1, Vgr-1, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6,GDF-7, GDF-8, GDF-9, and GDF-10, GDF-11, GDF-12, GDF-13, CDMP-3, UNIVIN,NODAL, SCREW, ADMP and NEURAL, and amino acid sequence variants thereof.In one currently preferred embodiment, useful osteogenic proteinsinclude any one of OP-1, OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6, BMP-9,and amino acid sequence variants and homologs thereof, including specieshomologs thereof.

In certain preferred embodiments, useful osteogenic proteins includethose having an amino acid sequence sharing at least 70% (e.g., at least80%) sequence homology or “similarity” with all or part of a naturallyoccurring reference morphogenic protein. A preferred reference proteinis human OP-1, and the reference sequence thereof is the C-terminalseven-cysteine domain present in osteogenically active forms of humanOP-1. This domain corresponds to residues 330431 of SEQ ID NO:2. Otherknown osteogenic proteins can also be used as a reference sequence. Inone embodiment, a candidate amino acid sequence can be aligned with areference amino acid sequence by using the method of Needleman et al.,J. Mol. Biol. 48:443-453 (1970), implemented conveniently by computerprograms such as the Align program (DNAstar, Inc.). Internal gaps andamino acid insertions in the candidate sequence are ignored for purposesof calculating the level of homology or identity between the candidateand reference sequences.

“Amino acid sequence homology” is understood herein to include bothamino acid sequence identity and similarity. Homologous sequences shareidentical and/or similar amino acid residues, where similar residues areconservative substitutions for, or “allowed point mutations” of,corresponding amino acid residues in an aligned reference sequence.Thus, a candidate polypeptide sequence that shares 70% amino acidhomology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Certain particularly preferred morphogenic polypeptides share at least60% (e.g., at least 65%) amino acid sequence identity with theC-terminal seven-cysteine domain of human OP-1.

As used herein, “conservative substitutions” are residues that arephysically or functionally similar to the corresponding referenceresidues. That is, a conservative substitution and its reference residuehave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like. Preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al. (1978), 5 Atlas of ProteinSequence and Structure, Suppl. 3, Ch. 22, pp. 354-352, Natl. Biomed.Res. Found., Washington, D.C. 20007. Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine. The term “conservative variant” or “conservative variation”also includes the use of a substituting amino acid residue in place ofan amino acid residue in a given parent amino acid sequence, whereantibodies specific for the parent sequence are also specific for, i.e.,“cross-react” or “immuno-react” with, the resulting substitutedpolypeptide sequence.

In other preferred embodiments, the family of osteogenic proteins usefulin the present invention are defined by a generic amino acid sequence.For example, Generic Sequence 7 (SEQ ID NO:4) and Generic Sequence 8(SEQ ID NO:5), disclosed below, accommodate the homologies shared amongpreferred protein family members identified to date, including OP-1,OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, 60A, DPP, Vg-1, Vgr-1,and GDF-1. The amino acid sequences for these proteins are describedherein and/or in the art. The generic sequences include the identicalamino acid residues shared by these sequences in the C-terminal six- orseven-cysteine skeletal domains (represented by Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequences. The generic sequences provide anappropriate cysteine skeleton where inter- or intra-molecular disulfidebonds can form. Those sequences contain certain specified amino acidsthat may influence the tertiary structure of the folded proteins. Inaddition, the generic sequences allow for an additional cysteine atposition 36 (Generic Sequence 7) or position 41 (Generic Sequence 8),thereby encompassing the biologically active sequences of OP-2 and OP-3.

GENERIC SEQUENCE 7 (SEQ ID NO: 4)           Leu Xaa Xaa Xaa Phe Xaa XaaXaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa          1                5                   10                 15 ProXaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa             20                 25                   30 Xaa Xaa Xaa XaaXaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa35                   40                  45                 50 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa        55                  60                  65                   70Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa                 75                 80                  85 Met Xaa ValXaa Xaa Cys Xaa Cys Xaa      90                  95wherein each Xaa is independently defined as follows (“Res.” means“residue”): Xaa at res.2=(Tyr or Lys); Xaa at res.3=(Val or Ile); Xaa atres.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaaat res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile); Xaa atres.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn orGlu); Xaa at res. 13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa atres.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res.9=(Gly or Ser); Xaa atres.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu orGly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr,Asp, Gln, Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa atres.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu orTyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thror Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly orLeu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly orPro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr);Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res.46=(Gln or Arg); Xaa atres.47=(Thr, Ala or Ser); Xaa at res.48 (Leu or Ile); Xaa at res.49=(Valor Met); Xaa at res.50=(Hs, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn,Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu);Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Seror Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa atres.57=(Val, Ala Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys,Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa at res.63=(Ala orVal); Xaa at res.65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg orGlu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp orGly); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val orLeu); Xaa at res.71=(Ser, Ala or Pro); Xaa at res.72=(Val, Leu, Met orIle); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His);Xaa at res.76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg orSer); Xaa at res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser,Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa atres.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg); Xaa atres.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu orHis); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trpor Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg, Lys,Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala); and Xaa at res.97 (His or Arg).

Generic Sequence 8 (SEQ ID NO:5) includes all of Generic Sequence 7 andin addition includes the following five amino acid at its N-terminus:Cys Xaa Xaa Xaa Xaa (SEQ ID NO:8), wherein Xaa at res.2=(Lys, Arg, Alaor Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln);and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid as defined as for Generic Sequence 7, with thedistinction that each residue number described for Generic Sequence 7 isshifted by five in Generic Sequence 8. For example, “Xaa at res.2=(Tyror Lys)” in Generic Sequence 7 corresponds to Xaa at res. 7 in GenericSequence 8.

In another embodiment, useful osteogenic proteins include thosecomprising sequences defined by Generic Sequences 9 (SEQ ID NO:6) and 10(SEQ ID NO:7). Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-9, human BMP10, human BMP-11, Drosophila 60A, Xenopus Vg-1, seaurchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6,human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouse GDF-3,human GDF-1, mouse GDF-1, chicken DORSALIN, DPP, Drosophila SCREW, mouseNODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10, humanGDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like Generic Sequence7, Generic Sequence 9 accommodates the C-terminal six-cysteine skeletonand, like Generic Sequence 8, Generic Sequence 10 accommodates theC-terminal seven-cysteine skeleton.

GENERIC SEQUENCE 9 (SEQ ID NO: 6) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa1               5                   10                  15 Pro Xaa XaaXaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa            20                  25                  30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa    35                  40                  45                  50 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa                55                  60                  65 Xaa Xaa XaaXaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa        70                  75                  80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys Xaa Cys Xaa85                  90                  95wherein each Xaa is independently defined as follows: Xaa at res.1=(Phe,Leu or Glu); Xaa at res.2=(Tyr, Phe, His, Arg, Thr, Lys, Gln, Val orGlu); Xaa at res.3=(Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu,Asn or Phe); Xaa at res.5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys,Ser, Glu, Ala or Asn); Xaa at res.7=(Asp, Glu, Leu, Ala or Gln); Xaa atres.8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaaat res. 10=(Trp or Met); Xaa at res. 11=(Gln, Leu, His, Glu, Asn, Asp,Ser or Gly); Xaa at res.12=(Asp, Asn, Ser, Lys, Arg, Glu or His); Xaa atres.13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile orVal); Xaa at res.16=(Ala, Ser, Tyr or Trp); Xaa at res.18=(Glu, Lys,Gln, Met, Pro, Leu, Arg, His or Lys); Xaa at res.19=(Gly, Glu, Asp, Lys,Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala,Ser, Gly, Met, Gln, His, Glu, Asp, Leu, Asn, Lys or Thr); Xaa atres.22=(Ala or Pro); Xaa at res.23=(Tyr, Phe, Asn, Ala or Arg); Xaa atres.24=(Tyr, His, Glu, Phe or Arg); Xaa at res.26=(Glu, Asp, Ala, Ser,Tyr, His, Lys, Arg, Gln or Gly); Xaa at res.28=(Glu, Asp, Leu, Val, Lys,Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp,Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa atres.32=(Pro, Ser, Ala or Val); Xaa at res.33=(Leu, Met, Glu, Phe orVal); Xaa at res.34 (Asn, Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa atres.35=(Ser, Ala, Glu, Asp, Thr, Leu, Lys, Gln or His); Xaa atres.36=(Tyr, His, Cys, Ile, Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa atres.37=(Met, Leu, Phe, Val, Gly or Tyr); Xaa at res.38=(Asn, Glu, Thr,Pro, Lys, His, Gly, Met, Val or Arg); Xaa at res.39=(Ala, Ser, Gly, Proor Phe); Xaa at res.40=(Thr, Ser, Leu, Pro, His or Met); Xaa atres.41=(Asn, Lys, Val, Thr or Gln); Xaa at res.42=(His, Tyr or Lys); Xaaat res.43=(Ala, Thr, Leu or Tyr); Xaa at res.44=(Ile, Thr, Val, Phe,Tyr, Met or Pro); Xaa at res.45=(Val, Leu, Met, Ile or His); Xaa atres.46=(Gln, Arg or Thr); Xaa at res.47=(Thr, Ser, Ala, Asn or His); Xaaat res.48=(Leu, Asn or Ile); Xaa at res.49=(Val, Met, Leu, Pro or Ile);Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res.51=(Phe, Leu,Ser, Asn, Met, Ala, Arg, Glu, Gly or Gln); Xaa at res.52=(Ile, Met, Leu,Val, Lys, Gln, Ala or Tyr); Xaa at res.53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res.54=(Pro, Asn, Ser, Val or Asp); Xaa atres.55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His); Xaa atres.56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaa atres.57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res.58=(Pro, Gly, Ser,Asp or Ala); Xaa at res.59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg or Gly);Xaa at res.60=(Pro, Ala, Val, Thr or Ser); Xaa at res.61=(Cys, Val orSer); Xaa at res.63=(Ala, Val or Thr); Xaa at res.65=(Thr, Ala, Glu,Val, Gly, Asp or Tyr); Xaa at res.66=(Gln, Lys, Glu, Arg or Val); Xaa atres.67=(Leu, Met, Thr or Tyr); Xaa at res.68=(Asn, Ser, Gly, Thr, Asp,Glu, Lys or Val); Xaa at res.69=(Ala, Pro, Gly or Ser); Xaa atres.70=(Ile, Thr, Leu or Val); Xaa at res.71=(Ser, Pro, Ala, Thr, Asn orGly); Xaa at res.72=(Val, Ile, Leu or Met); Xaa at res.74=(Tyr, Phe,Arg, Thr, Tyr or Met); Xaa at res.75=(Phe, Tyr, His, Leu, Ile, Lys, Glnor Val); Xaa at res.76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser,Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res.78=(Ser, Asn, Asp, Tyr,Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp,Val, Lys, Gly, Gln or Arg); Xaa at res.80=(Asn, Lys, Thr, Pro, Val, Ile,Arg, Ser or Gln); Xaa at res.81=(Val, Ile, Thr or Ala); Xaa atres.82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa at res.83=(Leu, Tyr,Lys or Ile); Xaa at res.84=(Lys, Arg, Asn, Tyr, Phe, Thr, Glu or Gly);Xaa at res.85=(Lys, Arg, His, Gln, Asn, Glu or Val); Xaa at res.86 (Tyr,His, Glu or Ile); Xaa at res.87=(Arg, Glu, Gln, Pro or Lys); Xaa atres.88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa at res.89=(Met or Ala); Xaaat res.90=(Val, Ile, Ala, Thr, Ser or Lys); Xaa at res.91=(Val or Ala);Xaa at res.92=(Arg, Lys, Gln, Asp, Glu, Val, Ala, Ser or Thr); Xaa atres.93=(Ala, Ser, Glu, Gly, Arg or Thr); Xaa at res.95=(Gly, Ala orThr); and Xaa at res.97=(His, Arg, Gly, Leu or Ser). Further, afterres.53 in rat BMP3b and mouse GDF-10 there is an Ile; after res.54 inGDF-1 there is a Thr; after res.54 in BMP3 there is a Val; after res.78in BMP-8 and DORSALIN there is a Gly; after res.37 in human GDF-1 thereare Pro, Gly, Gly, and Pro.

Generic Sequence 10. (SEQ ID NO:7) includes all of Generic Sequence 9and in addition includes the following five amino acid residues at itsN-terminus: Cys Xaa Xaa Xaa Xaa (SEQ ID NO:9), wherein Xaa atres.2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res.3=(Lys,Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa it res.4=(His, Gln, Arg, Lys,Thr, Leu, Val, Pro, or Tyr); and Xaa at res.5=(Gln, Thr, His, Arg, Pro,Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu). Accordingly, beginning atres.6, each “Xaa” in Generic Sequence 10 is a specified amino aciddefined as for Generic Sequence 9, with the distinction that eachresidue number described for Generic Sequence 9 is shifted by five inGeneric Sequence 10. For example, “Xaa at res.1=(Phe, Leu or Glu)” inGeneric Sequence 9 corresponds to Xaa at res.6 in Generic Sequence 10.

As noted above, certain preferred bone morphogenic proteins useful inthis invention have greater than 60%, preferably greater than 65%,identity with the C-terminal seven-cysteine domain of human OP-1. Theseparticularly preferred sequences include allelic and phylogeneticvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful proteins include active proteins comprising dimers having thegeneric amino acid sequence “OPX” (SEQ ID NO:3), which defines theseven-cysteine skeleton and accommodates the homologies between severalidentified variants of OP-1 and OP-2. Each Xaa in OPX is independentlyselected from the residues occurring at the corresponding position inthe C-terminal sequence of mouse or human OP-1 or OP-2.

OPX (SEQ ID NO: 3) Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp LeuGly Trp Xaa Asp Trp1               5                   10                  15 Xaa Ile AlaPro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                  25                  30                  35 LeuXaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa        40                  45                  50                  55Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala            60                  65                  70 Xaa Ser Val LeuTyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg75                  80                  85                  90 Asn MetVal Val Xaa Ala Cys Gly Cys His         95                  100wherein Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaa atres.11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ile orVal); Xaa at res.23=(Glu or Gln); Xaa at res.26=(Ala or Ser); Xaa atres.35=(Ala or Ser); Xaa at res.39=(Asn or Asp); Xaa at res.41=(Tyr orCys); Xaa at res.50=(Val or Leu); Xaa at res.52=(Ser or Thr); Xaa atres.56=(Phe or Leu); Xaa at res.57=(Ile or Met); Xaa at res.58=(Asn orLys); Xaa at res.60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val);Xaa at res.65=(Pro or Ala); Xaa at res.71=(Gln or Lys); Xaa atres.73=(Asn or Ser); Xaa at res.75=(Ile or Thr); Xaa at res.80=(Phe orTyr); Xaa at res.82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa atres.89=(Lys or Arg); Xaa at res.91=(Tyr or His); and Xaa at res.97=(Argor Lys).

In still another preferred embodiment, useful osteogenically activeproteins comprise an amino acid sequence encoded by a nucleic acid thathybridizes, under low, medium or high stringency hybridizationconditions, to DNA or RNA encoding reference osteogenic sequences.Exemplary reference sequences include the C-terminal sequences definingthe conserved seven-cysteine domains of OP-1, OP-2, BMP-2, BMP4, BMP-5,BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7, and the like. High stringenthybridization conditions are herein defined as hybridization in 40%formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C. Standardstringency conditions are well characterized in commercially available,standard molecular cloning texts. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook et al. (Cold Spring HarborLaboratory Press 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984).

The osteogenic proteins contemplated herein can be expressed from intactor truncated genomic DNA or cDNA, or from synthetic DNAs, in prokaryoticor eukaryotic host cells. The dimeric proteins can be isolated from theculture media and/or refolded and dimerized in vitro to formbiologically active compositions. Heterodimers can be formed in vitro bycombining separate, distinct polypeptide chains. Alternatively,heterodimers can be formed in a single cell by co-expressing nucleicacids encoding separate, distinct polypeptide chains. See, e.g., WO93/09229 and U.S. Pat. No. 5,411,941, for exemplary protocols forrecombinant heterodimer protein production. Currently preferred hostcells include, without limitation, prokaryotes including E. coli, andeukaryotes including yeast (e.g., Saccharomyces) or mammalian cells(e.g., CHO, COS or BSC cells). Other host cells can also be used toadvantage. Detailed descriptions of the proteins useful in the practiceof this invention, including how to make, use and test them forosteogenic activity, are disclosed in numerous publications, includingU.S. Pat. Nos. 5,266,683 and 5,011,691, the disclosures of which areincorporated by reference herein.

II. Formulation and Delivery Considerations

A. General Considerations

Devices and compositions of the invention can be formulated usingroutine methods. Useful formulation methodologies include lyophilizationof solubilized protein onto matrix or carrier materials. Useful proteinsolubilization solutions include ethanol, urea, physiological buffers(e.g., saline), acidic buffers, and acetonitrile/trifluoroacetic acidsolutions, and the like. See, for example, U.S. Pat. No. 5,266,683. Thedesired final concentration of protein will depend on the specificactivity of the protein as well as the type, volume, and anatomicallocation of the defect. In one preferred embodiment, useful proteinshave half maximal bone forming specific activity of 0.5-1.0 ngprotein/25 mg matrix. Proteins having lower specific activity may alsobe used. The desired final concentration of protein may depend on theage, sex and overall health of the recipient. For example, 10-1000 μgosteogenic protein per 4 cm² of defect is a generally effective dose.Smaller quantities may suffice for smaller defects or tears.Optimization of dosages requires no more than routine experimentationand is within the skill of the art.

A device of the invention can assume a variety of configurations. It cancomprise a synthetic or natural-sourced matrix configured in size andshape to fit the defect site to be repaired. Alternatively, the devicecan comprise a carrier to formulate a gel, paste, putty, cement, sheetor liquid. For example, a matrix-free osteogenic device in solution canbe formulated by solubilizing certain forms of OP-1 in acetate (20 mM,pH 4.5) or citrate buffers, or phosphate-buffered saline (pH 7.5). Insome instances, the osteogenic protein may not be entirely solubilizedand may precipitate upon administration into the defect locus.Suspensions, aggregate formation or in vivo precipitation does notimpair the operativeness of the matrix-free osteogenic device whenpracticed in accordance with the invention disclosed herein. Matrix-freedevices in liquid or semi-liquid forms are particularly suitable foradministration by injection, so as, to provide the device to a defectlocus by injection rather than surgical means. A series of matrix-freedevices is described below. Matrix materials, including particulatematerials, also can be added to these devices, to advantage.

In yet another embodiment of the present invention, the osteogenicdevice is prepared immediately prior to its delivery to the defectlocus. For example, carboxymethylcellulose (CMC) containing devices canbe prepared on-site, suitable for admixing immediately prior to surgery.In one embodiment, low viscosity CMC (AQUALON) is packaged andirradiated separately from the osteogenic protein OP-1. The OP-1 proteinthen is admixed with the CMC carrier, and tested for osteogenicactivity. Devices prepared in this manner are as biologically active asthe conventional device without CMC. The devices repair defect loci byinducing cartilage or tissue formation. The amount of osteogenic proteineffective for this purpose can be readily determined by one skilled inthe art.

B. Preparations of Bone Morphogenic Proteins

The following illustrates methods for preparing lyophilized OP-1. Otherlyophilized osteogenic proteins can be prepared in a similar manner.

OP-1 is lyophilized from 20 mM (pH 4.5) acetate buffer with 5% mannitol,lactose, glycine or other additive or bulking agent, using standardlyophilization protocols. OP-1 prepared in this manner can remainbiologically active for at least six months when stored at 4° C. to 30°C.

OP-1 can also be lyophilized from a succinate or citrate buffer (orother non-volatile buffer) for re-constitution in water, or from waterfor re-constitution in 20 mM (pH 4.5) acetate buffer. Generally,additives such as lactose, sucrose, glycine and mannitol are suitablefor use in lyophilized matrix-free osteogenic devices. In certainembodiments, such devices (0.5 mg/ml OP-1 and 5% additive) can beprepared in a wet or dry configuration prior to lyophilization.

For example, liquid formulations of OP-1 in 10 and 20 mM acetate buffer(pH 4, 4.5 and 5) with and without mannitol (0%, 1% and 5%) are stableand osteogenically active for at least six months.

III. Bioassays

An art-recognized bioassay for bone induction is described in Sampath etal., Proc. Natl. Acad. Sci. USA 80:6591-6595 (1983) and U.S. Pat. No.4,968,590, incorporated by reference herein. The assay entailsdepositing test samples in subcutaneous sites in recipient rats underether anesthesia. A 1 cm vertical incision is made under sterileconditions in the skin over the thoracic region, and a pocket isprepared by blunt dissection. In certain circumstances, approximately 25mg of the test sample is implanted deep into the pocket and the incisionis closed with a metallic skin clip. The heterotropic site allows forthe study of bone induction without the possible ambiguities resultingfrom the use of orthotropic sites.

The sequential cellular reactions occurring at the heterotropic site arecomplex. The multi-step cascade of endochondral bone formation includes:binding of fibrin and fibronectin to implanted matrix, chemotaxis ofcells, proliferation of fibroblasts, differentiation into chondroblasts,cartilage formation, vascular invasion, bone formation, remodeling, andbone marrow differentiation.

Successful implants exhibit a controlled progression through the variousstages of induced endochondral bone development, which include: (1)transient infiltration by polymorphonuclear leukocytes on day one; (2)mesenchymal cell migration and proliferation on days two and three; (3)chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartilage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoblastic and bone remodelingon days twelve to eighteen; and (8) hematopoietic bone marrowdifferentiation in the ossicle on day twenty-one.

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Staining with toluidine blue orhemotoxylin/eosin clearly demonstrates the ultimate development ofendochondral bone. A twelve-day bioassay is sufficient to determinewhether bone inducing activity is associated with the test sample.

Additionally, alkaline phosphatase activity can be used as a marker forosteogenesis. The enzyme activity can be determinedspectrophotometrically after homogenization of the excised testmaterial. The activity peaks at 9-10 days in vivo and thereafter slowlydeclines. Samples showing no bone development by histology should haveno alkaline phosphatase activity under these assay conditions. The assayis useful for quantifying bone formation shortly after the test samplesare removed from the rat. For example, samples containing osteogenicprotein at several levels of purity have been tested to determine themost effective dose/purity level, in order to seek a formulation thatcan be produced on an industrial scale. The results as measured byalkaline phosphatase activity level and histological evaluation can berepresented as “bone forming units.” One bone forming unit representsthe amount of protein required for half maximal bone forming activity onday 12. Additionally, dose curves can be constructed for bone inducingactivity in vivo at each step of a purification scheme by assaying theprotein concentration obtained at the step. Construction of such curvesrequire only routine experimentation.

IV. EXAMPLES

The following examples are meant to illustrate the methods and materialsof the present invention. Suitable modifications and adaptations of thedescribed conditions and parameters normally encountered in the artwhich are obvious to those skilled in the art are within the spirit andscope of this invention.

Example 1

This example demonstrates the efficacy of osteogenic protein inregenerating functional replacement laryngeal tissue in a canine(beagle) model.

To prepare an osteogenic device, donor canine thyroid lamina, which wasto be used as an allograft matrix, was frozen and thawed several timesto release and remove cells. The thyroid lamina was then demineralizedin 0.5 N HCl (e.g., four exchanges of 10 volumes of solution; 2 hoursper exchange). A 4.5 cm² piece of treated thyroid allograft matrix wascoated with about 250 μg mature OP-1 to form an implantable osteogenicdevice.

Surgery was performed using standard procedures. A 2 cm² defect ascreated in the left lamina of the thyroid cartilage of the host animalafter careful preparation of the perichondrium. The implant was adjustedto fit the defect and incorporated with several stitches. Theperichondrium and adjacent muscles then were replaced. Followingrecovery, the animal was allowed unrestricted motion. The animal wassacrificed at 18 postoperative weeks.

Prior to sacrifice, the healing progress was monitored visually and bypalpation. Surgery and recovery did not result in loss of voice. Manualmanipulation identified no gross abnormality and suggested that pliant,mechanically acceptable tissue had formed. Following sacrifice, theentire larynx was dissected and fixed in 4% paraformaldehyde andpost-fixed in 70% ethanol. Careful dissection of all soft tissuesidentified no ossification or pathological structures such aspathological mineralization, aberrant vascularization and the like. Thethyroid cartilage was well shaped on both the operated and unoperatedsides and it was difficult to indicate the operated side. The thyroidcartilage was of cartilage-like color with no appearance of increasedvascularization. By palpation only a slight protrusion of about 2 mmcould be found at the reconstructed side. Maximal finger pressureindicated no instability of the replacement cartilage. The new tissuewas similar in strength, flexibility and pliability to the originaltissue. No interference with laryngeal rays of motion was educed.

Histological analysis indicated good incorporation of the newly formedcartilage and bone into the defect area. The new cartilage tissueappeared to be permanent, i.e., stable, and not subject to resorption asevidenced by its continued existence at 4 months post-operation.Temporary cartilage typically is resorbed or converted to fibrotictissue within 3 months. Osteoblasts and new bone formation wereidentified in portions of the tissue, indicating the occurrence ofosteogenesis.

These data demonstrated that thyroid cartilage allograft pre-coated withOP-1 induced the formation of a mechanically acceptable reconstructionof a surgically created thyroid cartilage defect. A cartilaginouscarrier was shown to be acceptable with no rapid release to adjacentstructures. Thus, OP-1 can be used to stimulate cartilage growth andrepair.

Example 2

This example provides a protocol for determining the efficacy ofosteogenic protein in repairing large laryngeal tissue defects and forcomparative measurement of alternative carriers and osteogenic proteinconcentrations.

Here, a range of protein concentrations, i.e., 100 μg-500 μg, and twodifferent surgical protocols, i.e., devices implanted underperichondrium and devices implanted under fascia, are tested. It iscontemplated that fascia provides fewer progenitor cells thanperichondrium. Animals are sacrificed at 16 weeks. Table I belowsummarizes the protocol.

TABLE I Experiment Protocol Group Dogs Defect Implant Duration I 3Partial Control cartilage 4 months (⅔ of the right side of under the thethyroid cartilage) perichondrium II 3 Partial 100 μg OP-1 + 4 months (⅔of the right side of cartilage under the the thyroid cartilage)perichondrium III 3 Partial 500 μg OP-1 + 4 months (⅔ of the right sideof cartilage under the the thyroid cartilage) perichondrium IV 3 Partial500 μg OP-1 + 4 months (⅔ of the right side of cartilage under the thethyroid cartilage) fascia V 3 Partial 500 μg OP-1 + 4 months (⅔ of theright side of GuHCl-extracted the thyroid cartilage) cartilage under theperichondrium

Example 3

This example provides a protocol for determining the efficacy of asynthetic matrix in repairing laryngeal tissue in a mammal.

Cleft defects are surgically created in ⅔ of one side of the thyroidcartilage in the test animal. The defect sizes range from 2.0-4.5 cm².To prepare an osteogenic device, one of the following three types ofmatrices/carriers is used: (i) bone collagen matrix that has beendemineralized, gaudier-extracted, and combined with CMC, e.g., 0.15-0.25g of CMC/g polymer, to maximize conciseness, integrity and handlingproperties; (ii) synthetic collagen-GAG matrix; and (iii) matrix-freecarriers. The amount of osteogenic protein is (i) 50 μg/defect; (ii) 100μg/defect; (iii) 500 μg/defect; or (iv) 750 μg/defect. The surgeryprotocol for implanting the osteogenic device involves replacement ofperichondrium, or removal of perichondrium and replacement of fascia andmuscles only.

Animals are treated as described in Example 1. Animals are sacrificed at12 weeks, 18 weeks, 24 weeks and 36 weeks.

The mechanical integrity of the target tissue can be evaluated usingstandard protocols for measuring load-bearing capacity, range of motion,compressive strength, and the like.

It is anticipated that all matrix components will result in mechanicallyacceptable replacement tissue formation and that at 24 weeks or 36weeks, histology will reveal stable cartilage formation.

Example 4

This example provides a protocol for determining the efficacy ofosteogenic protein in regenerating mechanically acceptable, functionalreplacement larynx following partial or complete laryngoctomy.

Here, a defect sufficient to remove at least ⅔ of the larynx andinvolving multiple laryngeal ligaments and cartilages is created. Areplacement allograft matrix is created, using, for example, theprotocol described in Example 1 or 2, or the protocol described in PCTpublication WO 95/33502.

The replacement matrix is coated with osteogenic protein as describedabove (e.g., 10-1000 μg OP-1) and surgically implanted. Animals aremonitored visually and by manual manipulation, and sacrificed at 12weeks, 18 weeks, 24 weeks and 36 weeks post-operation. It is anticipatedthat full incorporation of the graft will result in the formation ofmechanically acceptable functional replacement cartilage and ligamenttissue, and that the replacement tissue will give rise to a flexibleopen structure without substantial loss of voice or sphincter activity.

Example 5

The efficacy of OP-1 in regeneration of dog larynx was examined bytreating thyroid cartilage defects with thyroid allografts covered withhost perichondrium. Prior to implantation, allografts were frozen,thawed and demineralized. Animals were sacrificed 4 months followingsurgery. Macroscopic examination of all specimens was done by alaryngeal surgeon and no pathological changes was observed in any of theneck areas of the sacrificed animal. No pathological ossification wasfound in surrounding muscles and other connective structures. Also, nochanges were found in the inner part of the larynx itself, including allthree laryngeal compartments, namely, vestibulum, cavum laryngis andcavum infraglotticum.

Eleven specimens from 11 animals were analyzed, including: (i) 3specimens from control dogs implanted only with allografts (Group I),(ii) 4 specimens from dogs whose implants were coated with 500 μg OP-1and placed under the perichondrium of the host thyroid cartilage (GroupII); (iii) 2 specimens from dogs whose implants were coated with 500 μgOP-1 and placed under the neck fascia of the host thyroid cartilage(Group III); and (iv) 2 specimens from dogs whose allograft implants hadbeen extracted with salt and guanidinium hydrochloride, coated with 500μg OP-1 and placed under the perichondrium of the host thyroid lamina(Group IV).

Upon termination, larynx and the surrounding structures were removed,inspected and fixed in 10% formalin for 48 hours. Then the left thyroidlamina containing the repaired defect was dissected out and post-fixedin 4% paraformaldehyde. Each thyroid lamina was divided into 4 blockscovering the entire specimen and each block was individually embeddedinto plastic. Histological analysis using serial sections throughout thedefect that were separated by approximately 1-2 mm was then performed.

Group I: Control Allograft Implants

In this control group, dried implants were not exposed to any solutionprior to implantation. All the implants were sutured in a way that theedge was overlapping the host defect site by approximately 2 mm.

In none of the three control specimens was new bone or cartilageformation observed. Moreover, the entire allografts remained completelyintact with no apparent reduction in size. No resorption, newvascularization or inflammation was observed. In one dog the implantedallograft slipped laterally because of failed sutures and the defect wasclosed by irregular fibrous connective tissue.

Group II: Implants Coated with OP-1 and Covered with Host Perichondrium

In these dogs, the closed defects appeared hard and stable uponmechanical (finger) compression. It was not possible to shift the defectarea by intensive palpation, indicating that the implants resistedregular mechanical strains at the implantation site. These strainsincluded compression of soft tissues (muscles, fascias, etc.) duringswallowing, breathing, and barking. Histological analysis indicated thatOP-1 induced bone, cartilage and ligament-like repair of the thyroidcartilage defects. The implanted allograft was not completely resorbedwithin a 4 month observation time.

Healing at the largest diameter of the defect was particularly examined.On both ends of the large defect site, newly formed cartilage wasevident. The new cartilage spanned about. 40-50% of the defect area andwas completely fused to the host thyroid cartilage. The new cartilagewas hyahine cartilage with crossing elastic cartilage fibers.

Young cartilage was observed, accompanied by a definite graduation frommesenchymal cells to chondroblasts to chondrocytes. As mesenchymal cellsdifferentiated into chondroblasts, the latter cells deposited matrixcomponents around themselves, surrounding themselves in their ownsecretory products. As a result, a small lacuna was formed. Thechondroblasts resided within these spaces without any contact with othercells. The matrix was acidophilic. Maturation of the chondroblasts intochondrocytes was accompanied by cellular hypertrophy and a change fromlacunar shape to an ovoid or angular configuration.

The allograft matrix was found at a distance from the new cartilagelayers, being physically separated by a fibrous tissue layer. Noremodeling of the cartilage at the left defect site was observed withinthe 4 month observation period, indicating that the newly formedcartilage was stable for at least that period. The much smaller amountof cartilage at the right defect site was in contact with a bone layer.The middle and right parts of the defect comprised of the remainingallograft, newly formed bone, and ligament-like structures. The new boneoccupied about 20-25% of the defect area. The surfaces of newly formedtrabeculi were irregularly covered with active osteoblasts depositingthick osteoid seams. The entire defect site was tightly packed into theconnective fibrous tissue. The cartilage and bone surfaces were directlycovered with perichondrium and periosteum-like tissue that was highlycellular and vascularized containing the precursor cells.

The defect site was embraced from outside with a ligament-like layer ofregular fibrous connective tissue. The significant feature of thisconnective tissue was the orderly, parallel orientation of collagenousfibers. The fibrocytes delimited the extent of individual bundles,making this tissue a low cellular material. The nuclei of the cells andthe fibers had a site-dependent wavy appearance. Such connective tissueis the predominant type that forms tendons and ligaments. As larynxcontains ligaments, it is expected to have precursor cells in thisparticular microenvironment. Individual bundles of these highlyorganized fibers were held together by loose connective tissue, which isalso a characteristic of ligaments and tendons. In addition, reducedvascularity of this tissue was a further marker of the ligaments thataccount for variable regenerative ability in standard orthopaedicprocedures.

In one dog, the implant slipped slightly medially, but remained largelyin place. Bone that covered both allograft surfaces was in directcontact with the allograft at the anterior (outer) defect site and at adistance from the allograft at the posterior (inner) defect site.Endochondral bone formation was observed, evidenced by replacement of acartilage anlage with bone. The allograft split into two piecesseparated by a connective fibrous tissue layer. A thick fibrous layerseparated also the newly formed bone from the posterior allograft site.Newly formed bone comprised of trabeculi covered with osteoid seams andactive cuboid osteoblasts. This indicated that bone induction was notdependent on the rate of allograft removal and that the allograftcomprised of type II collagen did not direct the type of tissue formedat the regeneration site.

Group III: Implants Coated with OP-1 and Covered with Neck Fascia

The results in this group of these dogs indicated that using neckfascia, instead of the perichondrium, to cover the implant resulted in asignificant delay in new tissue induction and allograft removal.

Group IV: Implants Chemically Extracted, Coated with OP-1 and Coveredwith Perichondrium

In this group of dogs, the closed defects appeared hard and stable uponmechanical (finger) compression and could not be differentiated fromthose from the Group II animals. Histological analysis indicated thatOP-1 induced bone, cartilage and ligament-like repair of thyroidcartilage defects. As in the Group II animals, the process was notcompleted during a 4 months observation period. However, effectivehealing of the laryngeal tissue defects was observed.

Healing at the largest diameter of the defect was particularly examined.On both ends of the large defect site, newly formed bone and cartilagewere evident. Bone and cartilage occupied about 30-35% and 25-30%,respectively, of the full thickness defect area. The boundary betweenthe host thyroid cartilage and the new bone healed by the formation of abone continuum, while the boundary between the host thyroid cartilageand the new cartilage by a cartilage continuum. The bone continuumdescribed a complete fusion to the host thyroid cartilage by amicrocallus formation mechanism. Namely, adolescent host thyroidcartilage lamina might contain a bone layer covered with two hyalinecartilage layers; by creating a defect during the surgical procedure,the host bone was eventually damaged (fractured); installing the OP-1coated implant into the defect site induced bone healing by microcallusformation.

In all the specimens and tissue blocks tested, whenever there was boneresiding in the thyroid lamina, there was also bone formed at theadjacent defect boundary. This observation suggested that OP-1 attractedprecursor cells from the host bone marrow. In contrast, if there was noresiding bone in the host thyroid lamina, cartilage continuum developed,connecting the host thyroid to the remaining allograft and/orsurrounding ligament-like tissue. In such a way newly formed tissues andthe unresorbed allograft composed a very tight regenerating defect site.Newly formed bone extended to the middle of the defect and was localizedbetween unresorbed allograft pieces. It was filled with hematopoieticmarrow and fully mineralized. As in the Group II animals, newly formedligament-like structures were also observed, where ligament bundlesattached to the newly formed cartilage and bone.

These results indicated that 500 μg of OP-1 delivered via a thyroidallograft carrier induced regeneration and repair of thyroid laminacartilage defects, and that the new tissue met the animals' mechanicalneeds for swallowing, barking and breathing. The new tissue, whichincluded bone, cartilage and ligament-like structures, composed morethan 80% of the defect area.

The results further indicated that the healing depended in large part onOP-1 and the surrounding tissues which provided the various precursorcells. The tissue differentiation in the healing process did not appearto be carrier-dependent, for a Type II collagen carrier did not solelypromote new cartilage formation.

These results also suggested that the three types of newly formedtissues and their appendices, e.g., bone marrow, blood vessels, etc.,were tightly connected into a “bone-cartilage-ligament continuum” oftissues. Thus, it appeared that OP-1 served as a multiple tissuemorphogen in this specific microenvironment.

Finally, these results indicated that OP-1 was not merely an osteogenicmorphogen—it could also induce the formation of permanent cartilage andligament-like tissues.

Example 6

This example describes another study on the efficacy of osteogenicprotein in regenerating new tissue at a defect site. This studycontained five experimental groups that were divided into twosub-studies. Groups I-III compared the effects of different OP-1carriers on the repair of identical thyroid cartilage defects. Thetested carriers were CMC, CMC/blood paste, and HELISTAT® sponge (a TypeI collagen composition). Groups IV and V addressed different animalmodels and surgical methods, where larger defects as used in humanclinical practice were created and repaired by combinations of OP-1/CMCdevice, VICRYL surgical mesh, and PYROST (a bone mineral composition)rigid supports. These latter two groups were approximations of thecombined product and procedure envisioned for a clinical setting.Surgeries on Groups I-III were performed one or two months beforesurgeries on Groups IV and V. The experimental protocol is summarizedbelow in Table II.

TABLE II Dog Larynx Reconstruction Using OP-1 Group Dogs Defect OP-1Duration I 3 I A 4 months II 3 I B 4 months III 3 I C 4 months IV 3 II A6 months V 3 III A × 2 6 months Defect I: Partial removal of rightthyroid lamina. OP-1 was applied to the defect and contained betweenperichondrial layers adjacent to the thyroid cartilage. II: Partialvertical laryngoctomy. An OP-1/CMC device was implanted between layersof VICRYL mesh. The implant was fixed to PYROST rods, which positionedand shaped the implant. The implant was contained between a pharyngealmucosa flap (inside) and the perichondrium (outside). III: Extendedpartial vertical laryngoctomy. OP-1/CMC devices (2 per animal) wereimplanted and fixed as described for partial vertical laryngoctomy. OP-1A. OP-1/CMC device. B. OP-1 in CMC/blood paste. C. OP-1 applied toHELISTAT ®

Analysis of the treated laryngeal tissue indicated that all threeformulations (OP-1/CMC device, OP-1/CMC blood paste, OP-1/HELISTAT®)induced bone and cartilage formation at the defect site. Some implantswere partially integrated and others were fully integrated with existingcartilage surrounding the defect sites.

Example 7

Using the protocols described in Examples 1-3, the efficacy ofosteogenic protein in generating mechanically acceptable replacement oftracheal hyaline cartilage rings and the annular ligament isdemonstrated. A defect sufficient to remove at least ⅔ of one of theseveral allocating hyaline cartilage rings is created. Donor trachealallograft matrix is prepared as described above in Example 1. Asynthetic polymer matrix can also be used. Preferably, 10-750 μg OP-1 isused. The replacement matrix is coated with the osteogenic protein andsurgically implanted between two remaining rings using metal-miniplates.

Animals are monitored by tracheal endoscopy and by manual palpitation.They are sacrificed at 24 weeks following surgery. It is anticipatedthat full incorporation of the graft will result, and newly inducedligament-like membrane will form and connect the new ring with theneighboring tracheal rings, giving rise to a flexible open tube-likestructure with interrupted respiration.

Example 8

The following protocol may be used to determine whether a morphogen suchas OP-1 is effective in vivo in promoting regeneration of tissue torepair defects in intervertebral discs.

Intervertebral discs are aseptically harvested from mature dogs, trimmedof all adherent tissue, and devitalized as described in Example 1. Eachdisc is bisected in the coronal plane and 3 mm full-thickness circulardefects are made in each half. The discs are coated with the morphogenand surgically re-implanted. The discs are examined for the extent ofrepair at the defect sites at various time points after re-implantation.

Example 9

This example demonstrates the efficacy of osteogenic protein instimulating cartilage matrix repair by cells, specifically nucleuspulposus (“NP”) and annulus fibrosus (“AF”) cells, isolated fromintervertebral discs (“IVDs”).

In this example, lumbar discs were isolated from New Zealand Whiterabbits and NP tissue was separated from AF tissue by dissection. NP andAF cells were separately isolated from the two tissues by sequentialenzyme digestion and re-suspended in 1.2% low viscosity sterilealginate, which was then formed into beads by expression through a 22gauge needle into a 102 mM CaCl₂ solution. The beads were separatelycultured in DMEM/F-12 medium containing 10% fetal bovine serum (“FBS”),25 μg/ml ascorbate and 50 μg/ml gentamycin. The medium was changeddaily.

After 14 days, each culture was subdivided into three groups. The firstgroup was a control group cultured for 12 more days. The second andthird groups were subjected to chemo-nucleolysis for 2 hours by 0.1 U/mlchondroitinase ABC (“C-ABC”), which is commonly used to degrade thechondroitin sulfate and dermatan sulfate chains of proteoglycans(“PGs”). Proteoglycans are a necessary component of the extracellularmatrix of IVDs. Low levels of PGs are associated with degenerative discdisease. It is believed that reduced PG synthesis plays a contributoryrole in disc degeneration. The second and third groups were subsequentlycultured for 12 days, the second group in the presence of 200 ng/ml ofOP-1, the third group in the absence of OP-1.

Assays were performed on all three groups immediately after the C-ABCtreatment, and at 3, 6, 9, and 12 days afterwards. The rate of mitosiswas determined by measuring the amount of DNA using the Hoechst 33258dye and fluorometry. The amount of sulfated PG synthesis was measuredusing the DMMB dye assay described in Hauselmann et al., J. Cell Sci.107:17-27 (1994), the teachings of which are herein incorporated byreference.

The cells of the second group cultivated in the presence of OP-1re-established a matrix significantly richer in PGs than those of thethird group cultivated in the absence of OP-1, as well as the firstcontrol group. These results show the osteogenic protein can stimulategrowth of the extracellular matrix.

Example 10

This example demonstrates the efficacy of osteogenic protein instimulating cartilage matrix repair by cells, specifically NP and AFcells, isolated from IVDs.

In this example, lumbar discs were isolated from New Zealand Whiterabbits and NP tissue was separated from AF tissue by dissection. NP andAF cells were separately isolated from the two tissues by sequentialenzyme digestion and re-suspended in 1.2% low viscosity sterilealginate, which was then formed into beads. The cells were separatelycultured in DMEM/F-12 medium containing 10% FBS, with the medium beingchanged daily. After 7 days, each culture was subdivided into threegroups. The first group was a control group which was not treated withOP-1. The second and third groups were grown in the presence of OP-1 for72 hours, the second group being treated with 100 ng/ml of OP-1, and thethird group being treated with 200 ng/ml of OP-1. Radiolabeled³H-proline was added to the cultures for the last 4 hours of incubationwith OP-1. After the incubation, collagen was extracted from thecultures, and the rate of collagen production was determined bymeasuring ³H-proline's incorporation into the extracts. Collagenproduction is associated with growth and repair of cartilage matrix. Todetermine the rate of cell proliferation, the content of each group'sDNA was measured using Hoechst 33258 dye.

Osteogenic protein increased collagen production in both NP and AF cellcultures in a concentration-dependent manner. The third groupincorporated more radiolabel than the second group, which in turnincorporated more radiolabel than the first control group. Osteogenicprotein had a significant mitogenic effect at high concentrations, whichaccounts for some of the elevation in collagen production. Nonetheless,the rate of collagen synthesis was significantly increased even whenincreased cell proliferation is accounted for. These results suggestthat osteogenic protein stimulates the growth and repair ofextracellular matrix.

Example 11

This example illustrates the efficacy of osteogenic protein instimulating synthesis of cartilage matrix components (e.g., collagen andPGS) by cells, specifically NP and AF cells, isolated from IVDs.

In this example, lumbar discs were isolated from New Zealand Whiterabbits and NP tissue was separated from AF tissue by dissection. NP andAF cells were separately isolated from the two tissues by sequentialenzyme digestion and encapsulated in 1.2% low viscosity sterile alginatebeads as described in Chiba et al. Spine 22:2885 (1997), the teachingsof which are herein incorporated by reference.

The beads were separately cultured in DMEM/F-12 medium containing 10%FBS, with the medium being changed daily. After 7 days, each culture wassubdivided into three groups. The first group was a control group whichwas not treated with OP-1. The second and third groups were grown in thepresence of OP-1 for 72 hours, the second group being treated with 100ng/ml of OP-1, and the third group being treated with 200 ng/ml of OP-1.

To provide a marker for collagen synthesis, radiolabeled ³H-proline wasadded to the cultures for the last 16 hours of incubation with OP-1. Toprovide a marker for PG synthesis, radiolabeled ³⁵S-sulfate was added tothe cultures for the last-4 hours of incubation with OP-1. To provide amarker for cell proliferation, MTT was added to the cultures for thelast 60 minutes of incubation with OP-1. Assays were then performed onthe cell cultures to measure cell proliferation, PG synthesis andcollagen synthesis. Cell proliferation was assayed by lysing andcentrifuging the cells and measuring the absorbance of the supernatantat 550 nm, as described in Mossman, J. Immunol. Methods 65:55 (1984),the teachings of which are herein incorporated by reference. PGsynthesis was determining by measuring incorporation of ³⁵S into thematrix, as described in Mok et al., J. Biol. Chem. 269:33021 (1994), andMasuda et al., Anal. Biochem. 217:167 (1994), the teachings of which areherein incorporated by reference. Collagen synthesis was assayed bymeasuring incorporation of ³H-proline into the matrix, as described inHauselmann et al., supra.

The data showed that OP-1 elevated synthesis of both PG and collagen inboth NP and AF cultures in a concentration-dependent manner. The thirdgroup incorporated more of both kinds of radiolabel than the secondgroup, which in turn incorporated more of both kinds of radiolabel thanthe first control group.

Osteogenic protein had a significant mitogenic effect at highconcentrations, which accounted for some of the elevation in collagenand PG production. Nonetheless, the rate of collagen and PG synthesiswas significantly increased even when increased cell proliferation wasaccounted for. These results suggest that osteogenic protein stimulatesthe growth and repair of extracellular matrix.

Example 12

The in vivo effects of OP-1 on the repair of intervertebral discs arestudied in two rabbit models—one model involves stab-wounding of theannulus fibrosus, as described in Lipson et al., Spine 6:194 (1981), andthe other model involves intradiscal C-ABC injection, as described inKato et al., Clin. Orthop. 253:301 (1990).

Briefly, for the stab-wounding method, an incision will be made in theannulus fibrosus of New Zealand White rabbits. Each rabbit will have twodiscs treated: one disc treated with OP-1 and the other treated withsaline. For the intradiscal injection model, the lumbar discs of NewZealand White rabbits will be exposed and C-ABC in the presence andabsence of OP-1 will be injected into the intervertebral discs. Atvarying times following treatment, the rabbits will be euthanized andthe effects of OP-1 on the repair of the intervertebral disc space willbe evaluated by methods well known in the art. These methods includemagnetic resonance imaging, mechanical tests, histological analysis, andbiochemical studies of the various extracellular matrix components inthe repaired discs.

Example 13

This example describes another study on the regeneration of dog larynxwith OP-1 and different carriers.

In this study, three different osteogenic devices were used to deliverOP-1. They were the OP-1/CMC device, OP-1/CMC/blood paste, andOP-1/HELISTAT sponge. The blood paste device was prepared by mixing 160μl OP-1 at 5 mg/ml with 400 μl 20% CMC via a syringe connection,followed by addition of 240 μl freshly drawn autologous blood andcontinuous mixing. The final volume applied to the defect was 0.8 ml.The HELISTAT device was prepared by applying 225 μl OP-1 onto 6 mgHELISTAT sponge for every 2 cm² defect area.

Three different treatment methods were studied. In the first treatmentmethod, defects in the left thyroid cartilage lamina were created asdescribed above; OP-1 devices were applied to the defect areas andmaintained between perichondrial layers adjacent to the defect. In thesecond treatment method, partial vertical laryngoctomy was initiallyperformed, and the OP-1/HELISTAT device was implanted; immobilization ofthe reconstructed area was achieved with PYROST as described in Example6; the implant was placed between a pharyngeal mucosal flap (inside) andthe perichondrium (outside). The third treatment method involvedanterior cricoid split and luminal augmentation; in this method, theOP-1/HELISTAT device was implanted and immobilized with PYROST.

During the course of experiment, the test animals had no recordedbreathing, eating and barking problems. Dogs were killed four monthsfollowing surgery and all specimens, including large reconstructedareas, appeared hard upon palpation. Dissection of the larynx wasperformed, with special care not to disturb incompletely healed areas,if any. Specimens were cut and embedded into plastics as previouslydescribed.

Group I: OP-1/CMC

This group of animals were treated with the first treatment method,supra, using the OP-1/CMC device. Thyroid defects in all three dogshealed almost completely. Surprisingly, although CMC might have been tooliquidy, the newly induced tissue was nicely positioned within thedefect margins. This observation suggested that the closure with softtissue was successfully performed. This was also the first evidence thatCMC could serve as a carrier for OP-1. Moreover, although there was noevidence that OP-1 remained within CMC for a longer period of time beingprotected against proteolytic degradation, the newly induced bone waswell incorporated into the defect. Unlike the above dog study where OP-1applied with an allograft matrix could induce bone, cartilage andligament, this study showed that only bone and ligament were formed. Thenew bone was well connected to both cartilage ends and embraced by aligament-like soft tissue. Von Kossa staining indicated completemineralization of the new bone. Abundant bone marrow filled the ossiclealmost completely. Remnants of cartilage anlage were found. Bonesurfaces were covered with very active osteoblasts, which wereaccumulating a thick layer of osteoid along the bone surfaces. Thecortical bone outside the newly formed ossicle was undergoing intensiveremodeling, as indicated by intracortical bone remodeling units filledwith osteoclasts, osteoblasts and blood capillaries. At severalcartilage-bone boundaries, the process of endochondral bone formationwas still active, although the border between the two tissues was notclearly demarcated. This result indicated that a new layer of cartilagewhich formed between old cartilage and new bone would ossify in time,and that newly formed cartilage was only transiently present and thus,lacked the characteristics of a permanent tissue.

In the dog study described in Example 5, cartilage allografts were usedas carriers for OP-1; the newly formed cartilage was separated from thebone and appeared permanent. However, in this study, where a differentcarrier (CMC) was used and the tissue formation was not controlled bythe slow release of morphogen or guided by an extracellular matrixcarrier, osteogenesis prevailed over chondrogenesis. This resultsuggested that precursor cells recruited for tissue formation in boththe previous and present studies came from the same cellular pool, andthat the morphogen threshold in the presence of CMC promotedosteogenesis. In other words, the carrier material and the morphogencontained therein coordinately influenced the outcome of tissuedifferentiation. Further, in Example 5, the allograft carriers were notcompletely removed by resorption within the 4 month observation period.Here, where CMC carriers were used, the rate of the healing wassignificantly faster, for the entire defect area was closed and almostcompletely remodeled within the same period of time.

Group II: OP-1/CMC/Blood

This group of animals were treated with the first treatment method,supra, using the OP-1/CMC/Blood device. The defects in all dogs healedcompletely. As in the Group I dogs, bone and ligament tissues wereinduced, while no new cartilage was apparent. The newly formed tissueswere nicely positioned within the defect margins. Addition of blood toCMC seemed to have created more new bone that was undergoingintracortical bone remodeling. The remodeling resulted in islands of newbone marrow with broad osteoid seams. The new bone was well connected toboth cartilage ends and embraced by a ligament-like soft tissue. VonKossa staining indicated complete mineralization of the new bone. Bonesurfaces were covered with active osteoblasts accumulating a thick layerof osteoid along the surfaces. The margins where the old cartilage andthe new bone merged were sharply separated by a thin layer of wellorganized connective tissue. No signs of endochondral bone formationwere detected within the old cartilage, suggesting that the process ofossification was faster in defects treated with the OP-1/CMC/blooddevice than in defects treated with the OP-1/CMC device. The presence ofosteogenic precursors present in the blood could have accounted for thisdifference.

Group III: OP-1/HELISTAT

This group of animals were treated with the first treatment method,supra, using the OP-1/HELISTAT sponge device. The defects in all dogshealed completely by the formation of new bone. Unlike the Group I andII dogs, the Group III dogs contained less ligament-like tissue at thehealed defect sites. In one animal, the new tissue was nicely positionedwithin the margins and only a small amount protruded laterally. In otheranimals, the new tissue formed multiple layers; in one dog the newtissue was completely out of the defect frame, inducing bone formationin the adjacent area.

The abundance of ossification was determined by the size and positioningof the BELISTAT sponge. Margins of the new bone and the old cartilagewere separated by a thin fibrous layer. Small amounts of collagen fromthe HELISTAT sponge remained unresorbed. Dislocation of the sponge inone animal led to abundant bone formation outside the defect site. Theorientation of bone trabeculi followed the path of collagen fiberswithin the sponge, suggesting that the ossification was guided by thecarrier matrix to which the morphogen had been bound. The decrease inthe amount of ligament-like tissue observed in this group of animals waslikely due to the lesser ability of Type I collagen to attract ligamentprecursor cells.

Group IV: Partial Vertical Laryngoctomy

This group of animals were treated with the second treatment method,supra, using the OP-1/HELISTAT sponge device. The anterior half of theleft thyroid lamina and the surrounding soft tissues (ventricular andvocal folds) were surgically removed. Immobilization of thereconstructed area was performed with PYROST. The implant was placedbetween a pharyngeal mucosal flap (inside) and the perichondrium(outside). Regeneration of the larynx skeleton was still in progresswith bone filling in the removed thyroid cartilage, as of 4 monthspost-operation. The new bone was still undergoing remodeling andprovided a good scaffold for the larynx skeleton integrity. The gapbetween the vocal and thyroid cartilages was filled with unorganizedconnective tissue, allowing normal air flow.

Group V: Anterior Cricoid Split with Luminal Augmentation

This group of animals were treated with the third treatment method,supra, using the OP-1/HELISTAT sponge device. The anterior part of thecricoid arcus was transected and a lumen extension was created byexternal implantation of PYROST. The space between the cricoid ends wasfilled with the OP-1/HELISTAT device. The lumen remained extended whilethe PYROST was partially removed or powdered and integrated with the newbone. The central area was occupied by new bone that was undergoingactive remodeling. Surprisingly, minimal bone tissue was formed adjacentto the PYROST, which might have served as an affinity matrix for theOP-1 protein released from the adjacent HELISTAT sponge. In onespecimen, the new bone and PYROST-surrounded bone formed an extendedbone area that did not compromise the lumen diameter. No ligament-liketissue was formed, indicating the lack of precursor cells in thevicinity of the cricoid cartilage.

1-56. (canceled)
 57. A method for repairing a defect locus in a ligamentin a mammal comprising the step of providing to the defect locus acomposition comprising an osteogenic protein selected from the groupconsisting of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6,BMP-9, BMP-10, BMP-11, BMP-15, BMP-3B, DPP, Vg-1, Vgr-1, 60A protein,GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10 andGDF-11; and a biocompatible, bioresorbable carrier; thereby inducing theformation of functional replacement ligament tissue.
 58. The method ofclaim 57, wherein the osteogenic protein is selected from the groupconsisting of OP-1, GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, BMP-5 and BMP-6.59. The method of claim 57, wherein the osteogenic protein is OP-1. 60.The method of claim 57, wherein the carrier is selected from the groupconsisting of a liquid, a semi-liquid and a gel.
 61. The method of claim57, wherein the carrier is selected from the group consisting ofcartilage, calcium phosphate, carboxymethylcellulose and collagen. 62.An implantable device for repairing a defect in a nonarticular cartilagetissue of a mammal, the device comprising an osteogenic protein selectedfrom the group consisting of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4,BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-15, BMP-3B, DPP, Vg-1, Vgr-1,60A protein, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,GDF-10 and GDF-11; and a biocompatible, bioresorbable carrier.
 63. Thedevice of claim 62, wherein the osteogenic protein is selected from thegroup consisting of OP-1, GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, BMP-5 andBMP-6.
 64. The device of claim 62, wherein the osteogenic protein isOP-1.
 65. The device of claim 62, wherein the carrier is selected fromthe group consisting of a liquid, a semi-liquid and a gel.
 66. Thedevice of claim 62, wherein the carrier is selected from the groupconsisting of cartilage, calcium phosphate, carboxymethylcellulose andcollagen.
 67. An implantable device for repairing a defect in anonarticular cartilage tissue of a mammal, the device comprising anosteogenic protein, wherein the osteogenic protein comprises an aminoacid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 or SEQ ID NO:7; and a biocompatible, bioresorbable carrier.
 68. Thedevice of claim 67, wherein the osteogenic protein comprises an aminoacid sequence set forth in SEQ ID NO:3.
 69. The device of claim 67,wherein the osteogenic protein comprises an amino acid sequence setforth in SEQ ID NO:4.
 70. The device of claim 67, wherein the osteogenicprotein comprises an amino acid sequence set forth in SEQ ID NO:5. 71.The device of claim 67, wherein the osteogenic protein comprises anamino acid sequence set forth in SEQ ID NO:6.
 72. The device of claim67, wherein the osteogenic protein comprises an amino acid sequence setforth in SEQ ID NO:7.
 73. The device of claim 67, wherein the carrier isselected from the group consisting of a liquid, a semi-liquid and a gel.74. The device of claim 67, wherein the carrier is selected from thegroup consisting of cartilage, calcium phosphate, carboxymethylcelluloseand collagen.